Embryonic Development (Embryogenesis/Gastrulation/Transcription/Coupled Translation-Glycosylation) JOSEPH T

Home , Embryonic development, Invagination

Proc. Natl Acad. Sci. USA Vol. 80, pp. 1028-1032, February 1983 Developmental Biology

Regulation of sea urchin glycoprotein mRNAs during embryonic development (embryogenesis/gastrulation/transcription/coupled translation-glycosylation) JOSEPH T. Y. LAU AND WILLIAM J. LENNARZ Department of Physiological Chemistry, The Johns Hopkins University School of Medicin6,i725 N. Wolfe Street, Baltimore, Maryland 21205 Communicated by Daniel Nathans, November 8, 1982 ABSTRACT Gastrulation in sea urchin embryos is accom- which the oligosaccharide is preassembled, and the rate of dol- panied by a striking increase in the synthesis of N-linked glyco- ichol phosphorylation (7) are increased after fertilization. proteins, and inhibitors of this process block gastrulation. In this Presently, nothing is known about the mRNAs that code for report, the messages coding for N-glycosylatable proteins in the glycoproteins in sea urchin embryos. The synthesis of these developing embryo of the sea urchin, Strongylocentrotus purpur- messages and their mobilization into active polysomes may be atmu, were examined. Total mRNAand mRNA isolated from mem- important regulatory points in the activation of glycoprotein branes of the embryos at various stages of development were used synthesis during gastrulation. What is known is that the unfer- to program a cell-free translation/glycosylation system-prepared tilized sea urchin egg contains a vast store of mRNA, which is from rabbit reticulocyte lysate supplemented with dog pancreas microsomes. The glycosylated translation products were sepa- thought to contain all of the information necessary for the early rated from the nonglycosylated products by concanavalin A-agar- development of the embryo (8-11). After fertilization, the stored ose and analyzed by gel electrophoresis. The results indicate that maternal mRNA is activated by an unknown mechanism and then although the RNA derived from the membranes of gastrula-stage mobilized into the polysomes (10). Although synthesis of mRNA embryos contains messages coding fornumerous glycoproteins, only begins shortly after fertilization, it has been suggested on the trace amounts of glycoprotein messages are associated with mem- basis of experiments' with actinomycin D to inhibit RNA syn- branes at earlier stages of development. mRNAs coding for four thesis that gastrulation represents the first stage requiring de glycoproteins of Mrs 70,000, 65,000, 51,000, and 30,000 were ex- novo RNA synthesis (for a review, see ref. 12). amined, further in total RNA preparations from the developing Because glycoproteins apparently need to be synthesized in embryo. The data indicate that the messages coding for the gly- order for the embryos to gastrulate (2-4), it was of interest to coproteins of Mrs 65,000 and 51,000 are present also in the un- examine the mRNAs coding for these glycoproteins to deter- fertilized egg and in the pregastrulation embryo. Because these mine if the messages are synthesized de novo or are maternal two messages are not found associated with the membranes until in origin and stored until gastrulation. Earlier studies have es- *gastrula stage, it is likely that the-synthesis of these glycoproteins tablished that a cell-free translation system supplemented with duringgastrulation is regulated atthe translationallevel. The mes- dog pancreas microsomes faithfully translates and glycosylates sages coding for glycoproteins of Mrs 70,000 and 30,000, on the proteins when.presented with the appropriate mRNA (13-15). other-hand, are not detectable in the unfertilized egg and may be In the currentstudy, glycoproteins synthesized invitro with un- synthesized de novo by the embryos. Thus, the expression of these fractionated sea urchin embryo RNA as message have been sep- two glycoproteins during gastrulation is regulated at least in part arated from the nonglycosylated translation products by con- on the transcriptional level. On the basis of these findings, it ap- canavalin A (Con A)-agarose and analyzed by NaDodSO4 gel pears thatdifferent modes of regulation are used fordifferent gly- electrophoresis. In this manner, total and membrane-associated coproteins that are synthesized during gastrulation. mRNA at various stages of development have been assayed for the presence of messages coding for N-glycosylatable proteins. Gastrulation in the developing embryo is a major morphoge- Wefirstexamined membrane-associated RNAfrom gastrula-stage netic event that requires coordinated cell migration and differ- embryos, a fraction that should contain the actively translating entiation. In sea urchin embryos that have developed to the glycoprotein messages. This RNA was found to be capable of mesenchyme blastula stage, the gastrulation process begins with programming the cell-free synthesis of numerous glycopro- invagination of the ectodermal cell layer near the vegetal pole. teins. Examination ofmembrane-associated RNAfrom embryos Subsequent emergence of proliferating secondary mesenchy- -at earlier stages of development indicates that these messages mal cells and continued invagination eventually leads to the for- are not associated with the membranes until gastrulation. In ad- mation of the primitive gut tube (see ref. 1 for review). Previous dition, the evidence indicates that although two of the glyco- studies from this laboratory have demonstrated that tunica- protein messages are present in the unfertilized egg, two others mycin and compactin, drugs that inhibitNlinked glycosylation, may be synthesized de novo after fertilization. prevent gastrulation without affecting earlier stages of development (2-5). These observations suggest that gastrulation represents the first developmental stage that,requires newly syn- MATERIALS AND METHODS thesized N-linked glycoproteins. Consistent with these obser- Materials. Sea urchins (Strongylocentrotus purpuratus) were vations, in vivo experiments with [3H]mannose and [3H]glu- purchased from Pacific Biomarine Supply (Venice, CA). The cosamine reveal that the onset ofgastrulationis accompaniedby animals, gametes, and embryos were handled and maintained a dramatic increase in glycoprotein synthesis (2, 5, 6). In ad- as described (2). [3SIMethionine (100GCi/mmol; 1 Ci = 3.7 X dition, the level of dolichyl phosphate (5), the lipid carrier upon 1010 Bq) and D-[2-3H]mannose (18.4 Ci/mmol) were purchased from Amersham. Micrococcal nuclease-treated rabbit reticu- The publication costs of this-article were defrayed in partby page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: endo H, endo-pN-acetylglucosaminidase H; Con A, nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. concanavalin A. 1028 Downloaded by guest on September 23, 2021 Developmental Biology: Lau and Lennarz Proc. Natl. Acad. Sci. USA 80 (1983) 1029 locyte lysate cell-free translation system and Con A-agarose were Con A buffer was added to the beads, incubated for 30 min, and purchased from Bethesda Research Laboratories; endo-,J-N- the buffer was removed. This was repeated four times and the acetylglucosaminidase H (endo H) was from A. Trimble (New a-methylmannoside supernatant fractions were pooled. 2-Mer York State Department of Health, Albany, NY). Fresh dog pan- captoethanol (final concentration, 1%) and cytochrome c as car- creas was obtained from W. Mitzner (The Johns Hopkins Uni- rier protein (60 ug) were added, and the protein was precipi- versity School of Hygiene and Public Health, Baltimore, MD). tated at 20C with 10% trichloroacetic acid for 4 hr. The tri- Isolation of RNA. For the isolation of total cellular RNA, the chloroacetic acid-insoluble pellet was washed three times with embryos were sedimented and washed once in Millipore (0.45- 90% ice-cold acetone and resolubilized in gel electrophoresis ,um pore size) filtered sea water. Packed embryos (1-2 ml) were sample buffer. solubilized in 40 ml of a solution containing 4 M guanidine iso- Other Procedures. For digestion with endo H, samples were thiocyanate as described by Chirgwin et al. (16). The RNA was diluted 1:1 with 0.4% NaDodSO4/1% 2-mercaptoethanol/0.2 purified by centrifugation through a cushion of 5.7 M CsCl as M 2-[N-morpholino]ethanesulfonic acid, pH 5.6/150 units of described by Glisin et aL (17). For the isolation of polysomes and aprotinin per ml and incubated overnight at room temperature membrane-associated RNA, the embryos (Ito 2 ml) were washed in the presence of 8 milliunits of the enzyme per ml. One-di- twice with Millipore-filtered sea water, twice with sterile Ca2+- mensional 10% polyacrylamide/NaDodSO4 gel electrophoresis and Mg2+-free, artificial sea water, and homogenized in 10 ml was performed as described by Laemmli (20) except that the of 10 mM Tris HCI, pH 7.5/10 mM KCI/2.5 mM MgCl2 with degree of crosslinking was halved. Two-dimensional gel elec- 50 strokes of a Dounce homogenizer. The homogenate was ad- trophoresis was carried out as described by O'Farrell (21). justedto 0.6 M sucrose/50 mM Tris HCI, pH 7.5/25mM KCI/2.5 mM MgCl2, and the nuclei were removed by centrifugation at RESULTS 1,000 X g for 10 min. The supernatant was centrifuged at 40,000 x g for 30 min to obtain a membrane fraction and a soluble frac- In Vitro Synthesis of N-linked Glycoproteins Using Gas- tion. To prepare membrane-associated RNA, the membrane trula-Stage mRNA. Because previous studies from this labo- fraction was extracted by using the guanidine isothiocyanate ratory have established that N-linked glycoprotein synthesis is procedure described above. The 40,000 x g soluble fraction was initiated at the onset of gastrulation, we first studied cell-free brought to 0.1% (vol/vol) Nonidet P40, layered on top of a synthesis of glycoproteins using RNA from gastrula-stage em- cushion consisting of 1.6 M sucrose/50mM Tris HCI, pH 7.5/250 bryos (45 hr after fertilization). Initially, RNA isolated from a mM KCI/2.5 mM MgCl2, and centrifuged at 27,000 X g for 3 membrane fraction that would be expected to contain the gly- hr in a SW 28 rotor. The resulting free polysome pellet was used coprotein polysomal messages was used to program in vitro to prepare RNA by the guanidine isothiocyanate method (16, translation reactions. Translation in the absence of microsomes 17). produced a large number of polypeptides (Fig. 1, lane A), none Preparation of Dog Pancreas Microsomes. Microsomes were of which were glycosylated and recoverable by binding to Con prepared from fresh dog pancreas as described by Shields and A-agarose (lane B). In contrast, when translation was carried out Blobel (15). The microsomes were treated further with micro- in the presence of microsomes (Fig. 1, lane C), a subset of the coccal nuclease (18) and passed through Sepharose CL-2B col- many polypeptides was found to be glycosylated and to bind to umn equilibrated with 10 mM Hepes, pH 7.5/0.5 mM MgCl2/ Con A-agarose (lane D). 0.1 mM dithiothreitol (19). The microsomal fraction was diluted Several lines of evidence indicated that the polypeptides in to 24 A260 units/ml and frozen at -70°C in small aliquots. the subset are indeed glycosylated proteins programmed by Cell-Free Synthesis of Glycoproteins. Translation reactions embryo mRNA. First, when the proteins selected by binding to (final volume, 30 ,l) were performed as recommended by Be- Con A-agarose were treated with endo H to cleave the oligo- thesda Research Laboratories except that the reactions were for saccharide chains, a decrease in Mr of =1,500 was observed in 75 min at 29°C in the presence of 25 ,uCi of [3S]methionine and all of the major bands (Fig. 1, lane E). Second, when the total 13.2 A1l of reticulocyte lysate containing 144 mM KC1. For cou- proteins were treated with endo H prior to Con A-agarose bind- pled in vitro translation/glycosylation reactions, 2 ,ul of dog ing, no glycosylated protein could be recovered by subsequent pancreas microsomes (24 A260 units/ml) were included in each Con A-agarose binding (Fig. 1, lane F). Finally, the glycopro- 30 ,l immediately before the initiation of the reaction. Under teins synthesized in the coupled translation/glycosylation sys- these conditions, synthesis of glycosylated vesicular stomatitus tem (Fig. 1, lane D) are of sea urchin mRNA origin because when virus G protein and ovalbumin was readily detected. the RNAwas omitted, no glycosylated products were detectable Analysis of Cell-Free Synthesized Glycosylated Proteins. (Fig. 1, lane G). As is evident in lane D, mRNA derived from Because dog pancreas microsomes do not contain the enzymes membranes of 45-hr embryos encodes a number of glycopro- that catalyze posttranslational processing of oligosaccharides on teins with Mrs ranging from 30,000 to >200,000. The Mr 200,000 glycoproteins, theinvitro synthesized glycoproteins shouldonly glycoproteins were not translated reproducibly from one RNA contain high mannose-type chains that bind specifically to Con preparation to the next; therefore, efforts were concentrated on A. Based on this property, glycosylated proteins can be selected four glycoproteins: Gp7O, Gp65, Gp5l, and Gp3O of Mrs 70,000, from a mixture of translation products by the use of Con A-agar 65,000, 51,000, and 30,000, respectively. ose. In atypicalassay, 3,lof3.5%(wt/vol) NaDodSO4was added Because membrane and secretory proteins are known to be to each 30 ,ul of translation mixture and incubated at room tem- synthesized on membrane-bound polysomes, the distribution perature for 5 min. Con A-agarose (25 ,ul) and Con A buffer (155 of glycoprotein messages between the free polysomes and ,l; 0.5 M NaCl/0. 1% Triton X-100/0. 1% Nonidet P-40/1 mM membranes was examined. RNA was prepared from free poly- MnCl2/1 mM CaCl2/20 mM Hepes, pH 7.5/4.5kallikrein units somes and membranes of 45-hr embryos and used to program of aprotinin per ml) were added. The mixture was incubated at in vitro translation reactions. The total translation products syn- room temperature for 2 hr with occasional shaking to resuspend thesized by free, polysome-derived RNA and membrane-bound the Con A-agarose beads. The mixture was then centrifuged, RNA appeared surprisingly similar (Fig. 2, lanes A and C, re- and the supernatant was discarded. The Con A-agarose beads spectively), probably as the result of some trapping of the more were washed 10 times with 1-ml portions of Con A buffer. To abundant free polysomal messages in the membrane fraction. elute the glycoproteins, 100 A.lof 0.5 M a-methylmannoside in However, when glycoproteins in these total translation prod- Downloaded by guest on September 23, 2021 1030 Developmental Biology: Lau and Lennarz Proc. NatL Acad. Sci. USA 80 (1983) FREE BOUND

200 * 200-

*Gp70 92.5 * *Gp7O *Gp65 68 - .+.. * *Gp65 *Gp51 ., *Gp55 43* t...... ri *Gp3O *Gp3O 25.7* Mem - - + + + + + Id . t ConA - + - + + + + A B C 1) EndoH ---++ FIG. 2. Glycosylated polypeptides synthesized in vitro by RNA de- AC.B D E F rivedfromfree polysomes and from the membranefractionof gastrula- stage embryos. RNAs isolated from free polysomes (lanes A and B) and FIG. 1. Glycosylated polypeptides synthesized in vitro by mem- the membrane fraction (lanes C and D) of gastrula-stage embryos (45 brane-associated RNA isolated from gastrula-stage embryos. RNA iso- hr) were used to program cell-free translation with dog pancreas mi- lated from the membrane fraction of gastrula-stage embryos (45 hr) crosomes. Shown are the total translation products (lanes A and C) and was used to program in vitro translation experiments, and the trans- theproducts ofConA-agarose selection fromthetotal translation prod- lation products were analyzed on NaDodSO4 gel electrophoresis. Mrs ucts (lanes B and D). Equal amounts of radioactive total translation are shown x 10'3. Lanes: A, total translation products formed in the products were applied to Con A-agarose in this experiment and those absence of dog pancreas microsomes; B, products of Con A-agarose se- shown in Figs. 3 and 4. Mrs are shown x 10-3. lection of the total translation products made as in lane A; C, total translation products formed in the presence of dog pancreas microsomes; D, products of Con A-agarose selection of the total translation products made as in lane C; E, products of endo H digestion of the Con A-agarose-selected material made as inlane D; F, products obtained by Con A-agarose selection when the total translation products made as in lane C were treated with endo H before selection; G, Con A-agarose selected translation products synthesized with lysate anddog pancreas microsomes in the absence of exogenous RNA. Mem, dog pancreas microsomes. ucts were selected by Con A-agarose, it was clear that the translation products programmed with membrane-derived RNA contained many glycoproteins (Fig. 2, lane D), whereas those 92.5 * programmed with free polysomal RNA contained only trace amounts of glycoproteins (Fig. 2, lane B). These results indicate 68- ; Gp7O that polysomes containing the messages for the glycosylatable :*Gp65 proteins are preferentially associated with membranes. There- fore, in subsequent experiments, mRNA associated with the *Gp51 membrane fraction was used to assess the presence of glyco- 43* protein messages in polysomes. mRNA Coding for Glycosylatable Proteins During Devel- opment. To determine at what stages of embryonic develop- -*Gp3O ment the messages for the N-linked glycoproteins first become 25.7- associated with the membrane fraction, RNA was prepared from the membrane fractions of embryos at various stages and used to program in vitro translation/glycosylation. Analysis of the - Nwrf Con A-agarose-selected translation products (Fig. 3) revealed that the RNA from membranes of early (12 hr) and hatched (24 FIG. 3. Glycosylated polypeptides synthesized in vitro by mem- hr) blastula-stage embryos were essentially devoid of messages brane-associated RNA isolated at different stages of development. the Membranes were prepared from embryos harvested at the times in- for glycoproteins. However, by early gastrula stage (32 hr), dicated after fertilization. The RNA prepared from these membranes messages for Gp7O, Gp65, and Gp5l were clearly evident. was used to program in vitro translations in the presence of dog pan- However, the message for Gp3O did not become associated with creas microsomes. Shown are the products obtained after Con A-agar- the membranes until the midgastrula stage (45 hr). ose selection of glycoproteins from the total translation products. Mrs The association of glycoprotein messages with membranes are shown x 10-3. Downloaded by guest on September 23, 2021 Developmental Biology: Lau and Lennarz Proc. Natl. Acad. Sci. USA 80 (1983) 1031 may be the result either of de novo synthesis of mRNA or of the messages for Gp65 and Gp51 were present and, therefore, of mobilization of mRNA from a stored pool. To distinguish be- maternal origin. In contrast, the products of the messages for tween these possibilities, total cellular RNA was isolated from Gp7O and Gp3O were absent, suggesting that these messages embryos at various stages of development and tested for its abil- may be synthesized de novo after fertilization. ity to direct in vitro glycoprotein synthesis. Electrophoretic profiles of the glycoproteins synthesized by total RNA isolated DISCUSSION from egg (0 hr), early blastula (12 hr), hatched blastula (24 hr), midgastrula (45 hr), and pluteus (72 hr) showed (Fig. 4) that some Gastrulation in sea urchin embryos is a complex morphogenetic mRNAs coding for N-glycosylatable proteins were present in event in which the participation of N-linked glycoproteins, per- the unfertilized egg. Gp65 and Gp51 were synthesized in vitro haps located on the cell surface, have been implicated. Earlier by the egg RNA, albeit at levels lower than seen by using mes- studies from our laboratory have demonstrated that the onset of sage from 24-hr embryos. In contrast, the egg appeared to be gastrulation is accompanied by initiation of N-linked glycopro- devoid of messages coding for Gp7O and Gp3O. By the hatched tein synthesis for the first time during development (2, 5, 6) and blastula stage (24 hr), the mRNAs encoding Gp7O, Gp65, and an enhancement of the dolichol-mediated N-glycosylation ap- Gp51 were clearlypresent. In addition, amessage foraMr 110,000 paratus (6). Consistent with these observations, we demon- glycoprotein that appears in abundance only at this stage of de- strated in the current study that mRNAs coding for glycopro- velopment was evident. The message for Gp3O probably was not teinsfirstbecome associated with membranes duringgastrulation. present during this stage of development, although a glycopro- The fact that much lower amounts of glycoprotein messages are tein with a slightly slower electrophoretic mobility than Gp3O found associated with membranes in embryos at earlier stages was synthesized. Gastrula-stage embryos (45 hr) contained of development strongly suggests that the availability of active mRNAs encoding Gp7O, Gp65, Gp51, and Gp3O, although the mRNA is a major factor in the initiation of glycoprotein syn- amount of Gp65 synthesized was much less than at earlier stages thesis. of development. By pluteus stage (72 hr), Gp3O was the pre- Based on whatwas known from studies on other proteins, the dominant glycoprotein synthesized, although Gp7O and Gp65 supply of active mRNA coding for glycoproteins could be reg- could still be detected. ulated either by synthesis of new transcripts or by mobilization To determine conclusively which of the glycoproteins syn- of mRNA from a dormant pool. The unfertilized egg contains a thesized by embryos during gastrulation are encoded by mes- vast store ofmessages that are activated afterfertilization (9, 22). sages already present in the unfertilized egg, in vitro translated Even during development, a substantial portion of the cyto- glycoproteins directed by egg RNA were analyzed on a two-di- plasmic mRNA is still sequestered in free ribonucleoprotein mensional gel and compared to the glycosylated proteins syn- particles (22-25). It often has been suggested that the control thesized by membrane-associated RNA from gastrula stage. The of protein synthesis can be exerted by the specific selection of products of the gastrula stage messages for Gp7O, Gp65, Gp5l, mRNAs from free ribonucleoprotein particles into polysomes and Gp3O were readily detectable (Fig. 5B). Examination of the (23, 25, 26). Our results suggest that at least two of the major products of the mRNA from the egg (Fig. 5A) revealed that the glycoproteins are regulated in this manner. In vitro translation/glycosylation reactions programmed with mRNA derived from the unfertilized egg clearly show that the messages for Gp65 and Gp51 are of maternal origin. Although additional copies of these messages may be synthesized after fertilization, there must _ JIM __ be specific controls to repress the translation of the existing messages until needed. It has been proposed that mRNAs have different affinities for a limiting amount of initiation factor (27- I

r 200 A i:. 31). If this is the case, selective expression of mRNA would be '.1 1.J...1 based on the ability of different classes of mRNAs to compete for initiation (31). In sea urchin embryos, a lower capacity of Z'C, mRNA from free ribonucleoprotein particles to bind ribosomes

* j. -.." 7 92.5 .... 'i., as compared to mRNA from polysomes has been reported (24). In addition, there is evidence indicating that initiation is rate- 68o *Gp7O limiting in blastula- and prism-stage S. purpuratus embryos (32). *Gp65 Our results suggest that the mRNAs for the glycoproteins Gp7O *Gp51 and Gp3O, are synthesized de novo in the developing embryo. 43o. However, the possibility that these sequences may exist in the egg as unprocessed, untranslatable transcripts that can be pro- cessed by the developing embryo cannot be ruled out. Trans- *Gp30 latable mRNA coding for Gp7O is present by the hatched blastula stage (24 hr); perhaps this message is stored until translated 25.7_ during gastrulation. The message coding for Gp3O, on the other hand, appears in the embryos at about the same time it is found CP -r MM associated with the membrane fraction, suggesting a direct re- LUJ vi LO N cruitment of the new mRNA into polysomes. Thus, the translation of the various glycoprotein mRNAs appears to be regu- FIG. 4. Glycosylated polypeptides synthesized in vitro by total cel- lated in different ways, depending on the particular message. lular RNA isolated at different stages of development. Total cellular Whereas the in vivo expression of Gp65 and Gp5l appears to RNA isolated from embryos at the times indicated was used to program in vitro translation with dog pancreas microsomes, and the products be under translational control, expression of Gp3O appears to be were subjected to Con A-agarose selection. Shown are the Con A-se- regulated by synthesis of mature message. The mRNA for Gp7O lected products synthesized with message isolated at 0, 12, 24, 45, and may be regulated by both means. It is not clear why different 72 hr after fertilization. Mrs are shown x 10'. modes of regulation are utilized; however, it is evident that such Downloaded by guest on September 23, 2021 1032 Developmental Biology: Lau and Lennarz Proc. Natl. Acad. Sci. USA 80 (1983)

A B

*.:1 ..:.. /.p70 1Gp70

Op65 Gp 65 Gp 51 *p 51 Op 30

FIG. 5. Glycosylated polypeptides synthesized in vitro by total egg RNA (A) and RNA derived from membranes of gastrula-stage embryos (B). The products of in vitro translation with dog pancreas microsomes were selected by Con A-agarose and subjected to two-dimensional gel analysis. The horizontal dimension is the isoelectric focusing dimension (most basic to most acidic, from left to right), and the vertical dimension is based on molecular weight. Arrows point to the positions of Gp7O, Gp65, Gp5l, and Gp3O. Also shown for comparison are equivalent one-dimensional NaDodSO4 gel analyses of the same samples.

controls are operative and that induction of glycosylation at gas- 8. Nemer, M. (1967) in Progress in Nucleic Acid Research and Mo- trulation is not merely dependent on the availability of the gly- lecular Biology, eds. Davidson, J. N. & Cohn, W. E. (Academic, cosylation apparatus, i.e. the glycosyl transferases and dolichyl New York), Vol. 7, pp. 243-301. 9. Gross, K. W, Jacobs-Lorena, M., Baglioni, C. & Gross, P. R. (1973) phosphate. Proc. Natl Acad. Sci. USA 70, 2614-2618. It should be noted that the use of a cell-free translation/ 10. Humphreys, T. (1971) Dev. Biol 26, 201-208. glycosylation system to assess the presence of mRNA encoding 11. Golan, G. A., Klein, W. H., Davis, M. M., Wold, B. J., Britten, glycosylatable proteins is not without limitations. In an early R. J. & Davidson, E. H. (1976) Cell 7, 487-505. report on the cell-free synthesis of glycosylated ovalbumin, it 12. Davidson, E. H. (1976) Gene Activity in Early Development was found that a substantial fraction of the nascent chains failed (Academic, New York). 13. Katz, F. N., Rothman, J. E., Lingappa, V. R., Blobel, G. & Lod- to interact with the microsomes, and, hence, were not glycosyl- ish, H. F. (1977) Proc. Natl. Acad. Sci. USA 74, 3278-3282. ated (33). Furthermore, when such a system is supplied with 14. Lingappa, V. R., Lingappa, J. R., Prasad, R., Ebner, K. E. & mRNAs coding for more than one glycoprotein, the nascent chains Blobel, G. (1978) Proc. Nat. Acad. Sci. USA 75, 2338-2342. often synthesized may not truly reflect the abundance of its 15. Shields, D. & Blobel, G. (1978) J. Biol. Chem. 253, 3753-3756. mRNA. Nevertheless, determination of gross changes in the 16. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, abundance of glycoprotein messages, and particularly the as- W. J. (1979) Biochemistry 18, 5294-5299. 17. Glisin, V., Crkvenjakov, R. & Byus, C. (1974) Biochemistry 13, sessment of whether a particular message is present, are cer- 2633-2637. tainly within the capabilities of this approach. Efforts are cur- 18. Scheele, G., Jacoby, R. & Carne, T. (1980)J. Cell Biol. 87, 611- rently underway in our laboratory to generate cDNA probes for 628. some of the glycoprotein messages so that changes in the levels 19. Walter, P. & Blobel, G. (1980) Proc. Nati Acad. Sci. USA 77, 7112- of these mRNAs during embryonic development can be more 7116. accurately assessed. 20. Laemmli, U. K. (1970) Nature (London) 227, 1335-1339. 21. O'Farrell, P. H. (1975)J. Biol. Chem. 250, 4007-4021. We are especially indebted to Dr. Don Cleveland for his expert ad- 22. Young, E. M. & Raff, R. A. (1979) Dev. Biol 72, 24-40. vice. In addition, we thank Drs. Joe Welply, Steve Grant, and John 23. Dworkin, M. B. & Infante, A. A. (1976) Dev. Biol. 53, 73-90. Morrow for helpful discussions, Mrs. Betty Earles for technical assis- 24. Rudensey, L. M. & Infante, A. A. (1979) Biochemistry 18, 3056- tance, and Ms. Ann Fuhr for preparation of the manuscript. This work 3063. was by a grant (HD 12718) from the National Institutes of 25. Infante, A. A. & Heilmann, L. J. (1981) Biochemistry 20, 1-8. supported 26. Raff, R. A. (1980) J. Cell Biol. 4, 107-136. Health. 27. Lodish, H. F. (1971)J. Biol. Chem. 246, 7131-7138. 28. Kaempfer, R., Hollender, R., Soreq, H. & Nudel, U. (1979) Eur. 1. Gustafson, T. & Wolpert, L. (1967) BioL Rev. 42, 442-498. 1. Biochem. 94, 591-600. 2. Heifetz, A. & Lennarz, W. J. (1979)1. Bio1 Chem. 254, 6119-6127. 29. DiSegni, J., Rosen, H. & Kaempfer, R. (1979) Biochemistry 18, 3. Schneider, E. G., Nguyen, H. J. & Lennarz, W. J. (1978)J. Biol 2847-2854. Chem. 253, 2348-2355. 30. Rosen, H., DiSegni, G. & Kaempfer, R. (1982)J. Biol Chem. 257, 4. Carson, D. D. & Lennarz, W. J. (1979) Proc. NatL Acad. Sci. USA 946-952. 76, 5709-5713. 31. Walden, W. E., Godefroy-Colburn, T. & Thach, R. E. (1981) J. 5. Carson, D. D. & Lennarz, W. J. (1980) J. BioL Chem. 256, 4679- Biol. Chem. 256, 11739-11746. 4686. 32. Hille, M. B., Hall, D. C., Yablonka-Reuveni, Z., Danilchik, M. 6. Lennarz, W. J. (1983) in Crit. Rev. Biochem., in press. V. & Moon, R. T. (1981) Dev. Biol. 86, 241-249. 7. Rossignol, D. P., Lennarz, W. J. & Waechter, C. J. (1981)J. Biol. 33. Lingappa, V. R., Shields, D., Woo, S. L. C. & Blobel, G. (1978) Chem. 256, 10538-10542. J. Cell Biol 79, 567-572. Downloaded by guest on September 23, 2021